The RecA protein of E. coli, which is both a protease and a recombinase, has a central role in repair of damaged DNA. RecA normally requires damaged DNA for conversion to the protease state, and both the protease and the recombinase functions participate in repair of damaged DNA. The protease function of RecA involves three functional sites, namely an active site that recognizes and cleaves substrates and the effector-binding sites whose states determine whether or not the active site exists. One aim of this project is to determine the location and structure of the three sites of RecA protease function and to determine also how they are related to the sites required for recombinase function. A mutational approach will be used. Only mutations that enhance function or that alter substrate specificity will be used to locate the sites; mutations causing loss of function may not be specific. Mutations called recA(Tif) confer constitutive protease activity on RecA protein without the usual need for DNA-damaging agents, and do so by enhancing binding of the two effectors that activate RecA to the protease state. DNA sequence analysis of numerous new recA(Tif) mutations isolated on LambdarecA will be carried out to identify the regions of RecA polypeptide that comprise the two effector-binding sites of RecA protease function; the aim is also to see whether the structure of these sites can explain their observed functional coupling. In vitro studies of the effector-binding properties of the RecA proteins from very strong recA(Tif) mutants are planned to test whether RecA protease strength is determined only by effector-binding strength. To identify the regions of RecA polypeptide that comprise the active site, the plan is to do a DNA sequence analysis of new types of recA mutations that alter the substrate recognition properties of the active site. Some element of RecA protease function plays a direct role in mutagenesis separate from its indirect inducing role. Various new recA mutants will be tested to see whether the aspect of protease function required for this direct role is the same as that required for cleavage of known repressors. To determine if any aspect of recombinase function is required for mutagenesis, recA mutants that are protease-proficient, recombinase-deficient will be tested. These split-phenotype mutants will also be used to determine whether protease or recombinase function is required for RecA-dependent replication, transcription, and transposon excision.